An existing stable isotope tracer methodology was used to monitor the flux through the de novo pyrimidine pathway in L1210-invaded spleens in vivo. The results of this study suggest that the effect of acivicin on carbamoyl-phosphate synthetase may be transitory and that the major effect of acivicin is due to inhibition of CTP-synthetase in the intact animal. Methodology was developed using GC/MS techniques in combination with HPLC separations for quantifying isotopic abundances in both purine and pyrimidine bases and nucleosides. A computer model using linear algebra techniques was developed which allows us to interpret data obtained from multiple stable label experiments. Studies were initiated using this methodology to determine the relative importance of the de novo pyrimidine and salvage biosynthetic pathways in normal and tumorous tissues in intact animals. Feasability of the method was tested using 15N-alanine, a time study in liver and intestine that showed the ratio of synthesis was linear with time. 1.5% and 7.5% of the uracil nucleotide pools contained in the liver and intestine, respectively were formed during a 1 h infusion of 15N-alanine.